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Identifying the mechanism behind delayed ethanol-induced headache (DEIH), otherwise known as the hangover headache, may provide insight into the mechanisms behind common headache triggers. Acetate was previously shown to be the key ethanol metabolite behind DEIH in the recurrent inflammatory stimulation (IS) rat model of headache. The reversal of trigeminal sensitivity following ethanol exposure with caffeine previously suggested a role of adenosine in DEIH. To characterize this, behavioral analysis and measurement of brainstem adenosine and glutamate with microdialysis and HPLC was performed while pharmacologically manipulating adenosine signaling in the IS and Spontaneous Trigeminal Allodynia (STA) rat models of headache. Blocking adenosine A2A receptor activation with istradefylline or acetate transport into astrocytes with the monocarboxylate transporter competitive inhibitor, alpha-cyano-4-hydroxycinnamate (4-CIN), prevented acetate-induced trigeminal sensitivity. Blocking adenosine A1, A2B, and A3 receptor signaling did not prevent trigeminal sensitivity. Compared to control rats, IS rats had greater increases in extracellular adenosine and glutamate within the trigeminal nucleus caudalis (TNC) of the brainstem during local acetate perfusion. Blocking transport of acetate into astrocytes with 4-CIN prevented the increase in adenosine and glutamate. Blocking A2A receptor activation prevented the increase in extracellular glutamate, but not adenosine in the TNC. These data are the first to demonstrate the physiological consequence of acetate on adenosinergic systems within trigeminal pain by suggesting that acetate-induced trigeminal sensitivity in DEIH is mediated by adenosine A2A receptor activation which modulates extracellular glutamate levels in the TNC.Significance StatementIt is unknown how several common headache triggers induce headache pain. Since migraineurs are more sensitive to these triggers, studying the mechanisms behind their effects may reveal unique migraine pathophysiology. In this study, we explored the common headache trigger, ethanol, which migraineurs are particularly sensitive to. When ethanol is ingested, its quickly metabolized to acetaldehyde and subsequently into acetate. We find that acetate increases brainstem adenosine and causes trigeminal sensitivity, which is exacerbated in the rat headache model. Blocking either acetate uptake or adenosine signaling prevents trigeminal sensitivity and brainstem glutamatergic signaling, suggesting that adenosine is involved in the hangover headache and that differences in acetate metabolism may account for the increased sensitivity to ethanol in migraineurs.

Populations of neural stem cells (NSCs) reside in a number of defined niches in the adult central nervous system (CNS) where they continually give rise to mature cell types throughout life, including newly born neurons. In addition to the prototypical niches of the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus, novel stem cell niches that are also neurogenic have recently been identified in multiple midline structures, including circumventricular organs (CVOs) of the brain. These resident NSCs serve as a homeostatic source of new neurons and glial cells under intact physiological conditions. Importantly, they may also have the potential for reparative processes in pathological states such as traumatic spinal cord injury (SCI) and traumatic brain injury (TBI). As the response in these novel CVO stem cell niches has been characterized after stroke but not following SCI or TBI, we quantitatively assessed cell proliferation and the neuronal and glial lineage fate of resident NSCs in three CVO nuclei—area postrema (AP), median eminence (ME), and subfornical organ (SFO) —in rat models of cervical contusion-type SCI and controlled cortical impact (CCI)-induced TBI. Using bromodeoxyuridine (BrdU) labeling of proliferating cells, we find that TBI significantly enhanced proliferation in AP, ME, and SFO, whereas cervical SCI had no effects at early or chronic time-points post-injury. In addition, SCI did not alter NSC differentiation profile into doublecortin-positive neuroblasts, GFAP-expressing astrocytes, or Olig2-labeled cells of the oligodendrocyte lineage within AP, ME, or SFO at both time-points. In contrast, CCI induced a pronounced increase in Sox2- and doublecortin-labeled cells in the AP and Iba1-labeled microglia in the SFO. Lastly, plasma derived from CCI animals significantly increased NSC expansion in an in vitro neurosphere assay, whereas plasma from SCI animals did not exert such an effect, suggesting that signaling factors present in blood may be relevant to stimulating CVO niches after CNS injury and may explain the differential in vivo effects of SCI and TBI on the novel stem cell niches.

BackgroundThe blood-brain barrier (BBB) has been hypothesized to play a role in migraine since the late 1970s. Despite this, limited investigation of the BBB in migraine has been conducted. We used the inflammatory soup rat model of trigeminal allodynia, which closely mimics chronic migraine, to determine the impact of repeated dural inflammatory stimulation on BBB permeability.MethodsThe sodium fluorescein BBB permeability assay was used in multiple brain regions (trigeminal nucleus caudalis (TNC), periaqueductal grey, frontal cortex, sub-cortex, and cortex directly below the area of dural activation) during the episodic and chronic stages of repeated inflammatory dural stimulation. Glial activation was assessed in the TNC via GFAP and OX42 immunoreactivity. Minocycline was tested for its ability to prevent BBB disruption and trigeminal sensitivity.ResultsNo astrocyte or microglial activation was found during the episodic stage, but BBB permeability and trigeminal sensitivity were increased. Astrocyte and microglial activation, BBB permeability, and trigeminal sensitivity were increased during the chronic stage. These changes were only found in the TNC. Minocycline treatment prevented BBB permeability modulation and trigeminal sensitivity during the episodic and chronic stages.DiscussionModulation of BBB permeability occurs centrally within the TNC following repeated dural inflammatory stimulation and may play a role in migraine.

The pain-signaling molecules, nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP), are implicated in the pathophysiology of post-traumatic headache (PTH) as they are for migraine. This study assessed the changes of inducible NOS (iNOS) and its cellular source in the trigeminal pain circuit, as well as the relationship between iNOS and CGRP after controlled cortical impact (CCI) injury in mice. The effects of a CGRP antagonist (MK8825) and sumatriptan on iNOS messenger RNA (mRNA) and protein were compared to vehicle at 2 weeks postinjury. Changes in CGRP levels in the trigeminal nucleus caudalis (TNC) in iNOS knockouts with CCI were compared to wild-type (WT) mice at 3 days and 2 weeks post injury. Trigeminal allodynia and photosensitivity were measured. MK8825 and sumatriptan increased allodynic thresholds in CCI groups compared to vehicle (p

Epi-illuminescence intravital fluorescence microscopy has been employed to study leukocyte-endothelial interactions in a number of brain pathologies. Historically, dyes such as Rhodamine 6G have been injected intravenously. However, intravenous injections can predispose experimental animals to a multitude of complications and requires a high degree of technical skill. Here, we study the efficacy of injecting Rhodamine 6G into the peritoneum (IP) for the purpose of analyzing leukocyte-endothelial interactions through a cranial window during real time intravital microscopy. After examining the number of rolling and adherent leukocytes through a cranial window, we found no advantage to the intravenous injection (IV). Additionally, we tested blood from both routes of injection by flow cytometry to gain a very precise picture of the two methods. The two routes of administration failed to show any difference in the ability to detect cells. The study supports the notion that IP Rhodamine 6G works as efficaciously as IV and should be considered a viable alternative in experimental design for investigations employing intravital microscopy. Facilitated intravital studies will allow for more exploration into cerebral pathologies and allow for more rapid translation from the laboratory to the patient with less chance of experimental error from failed IV access.

Post-traumatic headache is the most common symptom of postconcussion syndrome and becomes a chronic neurological disorder in a substantial proportion of patients. This review provides a brief overview of the epidemiology of postconcussion headache, research models used to study this disorder, as well as the proposed mechanisms. An objective of this review is to enhance the understanding of how the endogenous cannabinoid system is essential for maintaining the balance of the CNS and regulating inflammation after injury, and in turn making the endocannabinoid system a potential modulator of the trigeminal response to concussion. The review describes the role of endocannabinoid modulation of pain and the potential for use of phytocannabinoids to treat pain, migraine and concussion.

Migraine is the third most prevalent disease on the planet, yet our understanding of its mechanisms and pathophysiology is surprisingly incomplete. Recent studies have built upon decades of evidence that adenosine, a purine nucleoside that can act as a neuromodulator, is involved in pain transmission and sensitization. Clinical evidence and rodent studies have suggested that adenosine signaling also plays a critical role in migraine headache. This is further supported by the widespread use of caffeine, an adenosine receptor antagonist, in several headache treatments. In this review, we highlight evidence that supports the involvement of adenosine signaling in different forms of headache, headache triggers, and basic headache physiology. This evidence supports adenosine A2A receptors as a critical adenosine receptor subtype involved in headache pain. Adenosine A2A receptor signaling may contribute to headache via the modulation of intracellular Cyclic adenosine monophosphate (cAMP) production or 5' AMP-activated protein kinase (AMPK) activity in neurons and glia to affect glutamatergic synaptic transmission within the brainstem. This evidence supports the further study of adenosine signaling in headache and potentially illuminates it as a novel therapeutic target for migraine.

Performance of high repetition tasks with or without force is associated with peripheral tissue inflammation, decreased nerve function and motor dysfunction. Here, we examined whether a low repetition task with negligible force (LRNF) produces fewer tissue and behavioral pathologies than previously observed with high repetition tasks using our rat model of repetitive motion injury (RMI). Thirty-seven rats were randomized into control or LRNF groups, the latter reaching and grasping a 45 mg food pellet at a rate of 3 reaches/min. This task was performed in 4, 0.5 5 h sessions with 1.5 5 h rest periods for 3 days/week for up to 12 weeks. Examination of distal median nerve, forelimb flexor tendons and bones for ED1-positive cells (macrophages and osteoclasts) revealed increases in nerve and bone in week 12. The nerve also contained increased TNF-alpha expressing cells in week 12. Examination of spinal cord dorsal horns revealed increased immunoexpression of Substance P in week 8 and neurokinin-1 in weeks 8 and 12 in the superficial lamina. Motor behavioral analyses showed no changes in reach rate across weeks, slightly reduced task duration (a measurement of voluntary task participation) in week 12, but significantly increased extra arm movement reversals during reaching in week 8. These extra movement reversals were corrections for missed food pellets during a reach. Thus, performance of even a low repetition, negligible force upper extremity task for 3 months can induce mild peripheral tissue inflammation, neurochemical increases in spinal cord dorsal horns, and declines in fine motor control.

Mitochondrial Ca2+ homeostasis controls intracellular calcium signals, cellular energy production and mechanisms of cell death. We and others have shown that MICU1 is an intermembrane space protein that provides Ca2+ sensitivity to Ca2+ uptake by the mitochondria through the mitochondrial Ca2+ uniporter. Specifically, MICU1 is required both for protecting mitochondria from Ca2+ uptake when the cytoplasmic [Ca2+] is low and for proper transmission of the short-lasting Ca2+ signal from the ER/SR to the mitochondria. Human loss of function mutation of MICU1 has been linked to learning difficulties, skeletal muscle weakness, fatigue and motoric impairment.In this study, we describe and validate a neuron-specific MICU1 KO model in mice. Proteomic analysis of the mitochondria confirms loss of MICU1 and indicates a possible compensatory mechanism for the loss of MICU1 by other constituents of the uniporter. Behavioral tests performed on the animals showed decreased motoric function progressing with age. Measurements of cytoplasmic and mitochondrial Ca2+ signals in cultured cortical neurons and Ca2+ uptake by isolated mitochondria revealed altered Ca2+ homeostasis which might underlie the functional impairments.Our results reveal the specific contributions of MICU1 in neuronal Ca2+ homeostasis and provide some clues to the neuronal pathogenesis in MICU1 loss of function patients.

Long-term disability from traumatic brain injury (TBI) is prevalent occurring in approximately 3.17 million persons on an annual basis.1 Among the long-term consequences of TBI, post-traumatic headache (PTH) disorder represents the most common chronic pain syndrome within this patient population.2–7 PTH disorders are highly prevalent across all grades of TBI severity.8 Mild and moderate TBI is more prevalent compared to severe cases of TBI; however, there is a current controversy surrounding epidemiologic reports that cite a higher incidence of PTH for mild TBI compared with moderate and severe cases. A recent prospective 12-month study of TBI patients provides compelling evidence that the prevalence of PTH is unrelated to injury severity.9 Important aspects of PTH to consider relate to the time-course of this disorder. In many patients, it resolves in 3 months; in others, it persists for much longer. Currently, PTH, as classified by the International Classification of Headache Disorders, is defined as headache secondary to head trauma that develops within 7 days after injury or regaining consciousness. TBI is a risk factor for acute episodic headache (headache occurring less than 15 days/month) persisting less than 3 months transitioning into chronic headache (≥15 days/month).10,11 Clinical evidence suggest that the persistence of PTH beyond the expected tissue-healing time-course would argue for its chronicity, as well as mechanisms of central sensitization.12–14 A recent prospective study found the onset of PTH occurred between 7 and 30 days after injury or later in nearly half of patients.15 A study by Ofek and Defrin found that patients with TBI had a mean onset of chronic pain, including head pain, just over 6 months after injury.14 Headache was reported at 3, 6, and 12 months in 41% of patients with TBI in a study by Hoffman and colleagues.9 Studies using quantitative sensory testing (eg, von Frey mechanical stimuli for allodynia [cutaneous sensitivity to mechanical stimuli that are innocuous under normal conditions]) show that 40% of patients with TBI experience chronic head and face pain after head injury.14,16 Significant reductions in pressure-pain thresholds were found at least 1 year after mild-to-moderate TBI.16 To date, investigations into the proposed mechanisms and treatments for PTH have been impeded by a lack of preclinical models. Therefore, a research initiative implemented by our laboratory is to study mechanisms of PTH and its chronification using a well-known animal model of TBI, controlled cortical impact (CCI) injury. Headache pain, whether acute or chronic, involves abnormal activation of the trigeminovascular system. Using a model of diffuse TBI, Hall and Lifshitz have characterized hypersensitivity of the whiskers to be associated with neuroplasticity in the cortical barrel circuit and thalamus.17 Peripheral axons of the trigeminal ganglion innervate the peri-orbital skin, facial whiskers, anterior scalp, meninges, and cerebral vasculature. Periorbital and facial allodynia have been well-documented in rodent migraine models following infusion of an inflammatory soup over the meninges.18–20 Afferent fibers of the trigeminal ganglia relay nociceptive information to the trigeminal nuclei in which the neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP), play important roles. CGRP and SP are also important in normal physiologic and pathologic function including cerebrovascular regulation and the development of neurogenic inflammation.21–26 CGRP has been particularly well-studied for its role in primary headache disorders such as migraine.27,28 To date, preclinical studies examining allodynia and the potential role of CGRP and SP in PTH are nonexistent. This study aimed to characterize periorbital sensory changes to mechanical stimuli (allodynia) and neuroplasticity in the brainstem in a mouse model of TBI, as this has not yet been reported in any model of TBI. The present study tests the hypothesis that focal injury to the somatosensory cortex will be associated with increased neuropeptides within the brainstem trigeminal nucleus caudalis and periorbital allodynia in a mouse model of CCI injury. The time-course for macrophage/microglial and astrocyte responses in the somatosensory cortex after CCI were examined because these cell populations are a potential cellular source of mediators (eg, cytokines, nitric oxide [NO], excitatory amino acids) of nociceptor sensitization known to contribute to behavioral morbidity. The pattern of periorbital allodynia over 4 weeks in mice with CCI and those undergoing a craniotomy only was characterized, while changes in the nociceptive neuropeptides, CGRP, and SP within the brainstem were determined. The temporal relationships between periorbital sensory changes (allodynia), central nociceptive neuropeptides, and gliosis (macrophage/microglia and astrocyte activation) were examined.

Proposed therapeutic strategies for attenuating secondary traumatic brain injury (TBI) include modulation of acute neuroimmune responses. The goal of this study was to examine the acute effects of cannabinoid-2 receptor (CB(2)R) modulation on behavioral deficits, cerebral edema, perivascular substance P, and macrophage/microglial activation in a murine model of TBI. Thirty male C57BL/6 mice underwent sham surgery, or cortical contusion impact injury (CCI). CCI mice received vehicle or the CB(2)R agonist 0-1966 at 1 and 24 h after injury. Performance on the rotarod, forelimb cylinder, and open-field tests were evaluated before and at 48 h after sham or CCI surgery. Cerebral edema was evaluated using the wet-dry weight technique. Immunohistochemical analysis was used to examine changes in substance P and macrophage/microglia-specific Iba1 protein immunoreactivity. Locomotor performance and exploratory behavior were significantly improved in mice receiving 0-1966 (CB(2)R agonist) compared to vehicle-treated mice. Significant reductions were found for cerebral edema, number of perivascular areas of substance P immunoreactivity, and number of activated macrophages/microglial cells in the injured brains of 0-1966-treated mice compared to vehicle-treated mice. The findings show that the effects of the CB(2)R agonist 0-1966 on edema, substance P immunoreactivity, and macrophage/microglial activation, were associated with recovery of acute motor and exploratory deficits. This study provides evidence of acute neuroprotective effects derived from selective CB(2)R activation that may represent an avenue for further development of novel therapeutic agents in the treatment of TBI.

We investigated inflammation in rats performing a low repetition, negligible force (LRNF) or high repetition, negligible force (HRNF) task of reaching and retrieving food pellets at target rates of two or four reaches/min for 2 h/day, for 6-8 weeks. Serum was assayed for 11 cytokines and chemokines; forelimb tissues for four cytokines. Macrophages were counted in forelimb tissues of LRNF rats to add to results from our previous studies of HRNF rats. In HRNF rats, serum IL-1a, IL-1b, TNFa, MIP2, MIP3a, and RANTES were elevated in weeks 6 and 8. In contrast, only MIP2 and MIP3a increased in serum of LRNF rats. In 8 week HRNF reach limb tissues, IL-1a, IL-1b, TNFa, and IL-10 increased in distal bones, IL-1a and -b in muscles, and TNFa in tendons. Only IL-10 increased in LRNF reach limb muscles in week 8. Serum IL-1a and MIP2 correlated with macrophages in LRNF loose connective tissues, serum MIP3a and MIP2 correlated negatively with grip strength, while serum TNFa, MIP3a, and MIP2 correlated positively with total number of reaches. Thus, several tissue and circulating cytokines/chemokines increase in an exposure dependent manner following short-term performance of repetitive reaching tasks and correlate with macrophage infiltration and decreasing grip strength. 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

The purpose of this study was to compare the effects of crystalloid and crystalloid-colloid solutions administered at different times after isolated traumatic brain injury. Male Sprague-Dawley rats were randomized to receive one of three intravenous treatments (4 mL/kg body weight) at 10 min or 6 h after moderate traumatic brain injury. Treatments included hypertonic saline, hypertonic albumin, and normal albumin. Moderate injuries were produced using the controlled cortical impact injury model set at 2.0 mm, 4.0 m/sec, and 130 msec. Tissue damage and cerebral edema were measured to evaluate the effect of treatments for traumatic brain injury. Blood brain barrier permeability was assessed at different time points after injury to identify a mechanism for treatment effectiveness. Injury volume was the smallest for animals treated with hypertonic albumin at 6 h after injury compared to all other treatments and administration times. Ipsilateral brain water content was significantly attenuated with immediate normal saline-albumin treatment. The presence of colloid in the infusion solutions was associated with an improvement in tissue damage and edema following isolated head injury while hypertonic saline alone, when given immediately after injury, worsened tissue damage and edema. When hypertonic saline was administered at 6 h after injury, tissue damage and edema were not worsened. In conclusion, the presence of colloid in solutions used to treat traumatic brain injury and the timing of treatment have a significant impact on tissue damage and edema.

Background Frequent mild head injuries or concussion along with the presence of headache may contribute to the persistence of concussion symptoms. Methods In this study, the acute effects of recovery between mild head injuries and the frequency of injuries on a headache behavior, trigeminal allodynia, was assessed using von Frey testing up to one week after injury, while histopathological changes in the trigeminal pain pathway were evaluated using western blot, ELISA and immunohistochemistry. Results A decreased recovery time combined with an increased mild closed head injury (CHI) frequency results in reduced trigeminal allodynia thresholds compared to controls. The repetitive CHI group with the highest injury frequency showed the greatest reduction in trigeminal thresholds along with greatest increased levels of calcitonin gene-related peptide (CGRP) in the trigeminal nucleus caudalis. Repetitive CHI resulted in astrogliosis in the central trigeminal system, increased GFAP protein levels in the sensory barrel cortex, and an increased number of microglia cells in the trigeminal nucleus caudalis. Conclusions Headache behavior in rats is dependent on the injury frequency and recovery interval between mild head injuries. A worsening of headache behavior after repetitive mild head injuries was concomitant with increases in CGRP levels, the presence of astrocytosis, and microglia proliferation in the central trigeminal pathway. Signaling between neurons and proliferating microglia in the trigeminal pain system may contribute to the initiation of acute headache after concussion or other traumatic brain injuries.

Background Immunomodulatory therapies have been identified as interventions for secondary injury after traumatic brain injury (TBI). The cannabinoid receptor type-2 (CB2R) is proposed to play an important, endogenous role in regulating inflammation. The effects of CB2R stimulation, blockade, and deletion on the neurovascular inflammatory responses to TBI were assessed. Methods Wild-type C57BL/6 or CB2R knockout mice were randomly assigned to controlled cortical impact (CCI) injury or to craniotomy control groups. The effects of treatment with synthetic, selective CB2R agonists (0-1966 and JWH-133), a selective CB2R antagonist, or vehicle solution administered to CCI groups were assessed at 1-day after injury. Changes in TNF-α, intracellular adhesion molecule (ICAM-1), inducible nitric oxide synthase (iNOS), macrophage/microglial ionized calcium-binding adaptor molecule, and blood-brain-barrier (BBB) permeability were assessed using ELISA, quantitative RT-PCR, immunohistochemistry, and fluorometric analysis of sodium fluorescein uptake. CB2R knockouts and wild-type mice with CCI injury were treated with a CB2R agonist or vehicle treatment. Results TNF-α mRNA increased at 6 hours and 1 to 3 days after CCI; a CB2R antagonist and genetic knockout of the CB2R exacerbated TNF-α mRNA expression. Treatment with a CB2R agonist attenuated TNF-α protein levels indicating post-transcriptional mechanisms. Intracellular adhesion molecule (ICAM-1) mRNA was increased at 6 hours, and at 1 to 2 days after CCI, reduced in mice treated with a CB2R agonist, and increased in CB2R knockout mice with CCI. Sodium fluorescein uptake was increased in CB2R knockouts after CCI, with and without a CB2R agonist. iNOS mRNA expression peaked early (6 hours) and remained increased from 1 to 3 days after injury. Treatment with a CB2R agonist attenuated increases in iNOS mRNA expression, while genetic deletion of the CB2R resulted in substantial increases in iNOS expression. Double label immunohistochemistry confirmed that iNOS was expressed by macrophage/microglia in the injured cortex. Conclusion Findings demonstrate that the endogenous cannabinoid system and CB2R play an important role in regulating inflammation and neurovascular responses in the traumatically injured brain. CB2R stimulation with two agonists (0-1966 and JWH-133) dampened post-traumatic inflammation, while blockade or deletion of the CB2R worsened inflammation. Findings support previous evidence that modulating the CB2R alters infiltrating macrophages and activated resident microglia. Further investigation into the role of the CB2R on specific immune cell populations in the injured brain is warranted.